Orally administrable polyvalent vaccines for intestinal infections

ABSTRACT

FOR PROPHYLACTIC IMMUNIZATION AGAINST A WIDE VARIETY OF LOCAL INTESTINAL INFECTIONS, E.G. SALMONELLAL ENTERITIS, THERE IS PROVIDED AN ORAL POLYVALENT VACCINE COMPOSITION CONTAINING A SYNERGISTIC MIXTURE OF DIFFERENT INACTIVATED VACCINE TYPES SPECIFIC AGAINST DIFFERENT GROUPS OF MICROORGANISMS.

United States Patent Office 3,651,214 Patented Mar. 21, 1972 U.S.'Cl. 424-92 Claims ABSTRACT OF THE DISCLOSURE -'=-"'For prophylactic immunization against a wide variety of local intestinal infections, e.g. salmonellal enteritis, there is provided an oral polyvalent vaccine composition containing a synergistic mixture of different inactivated vaccine types specific against different groups of microorganisms.

BACKGROUND OF THE INVENTION i This invention relates in general to vaccines, and in particular to orally administrable, polyvalent synergistic mixtures of vaccines especially applicable to the development ofimmunity against intestinal infections.

Among the infections of the intestine, there are basically two different clinical types, namely the cyclic diseases and the local diseases. The cyclic intestinal infections, e.g. typhus and paratyphoid, characterized by a course of different phases, namely normal incubating period, generalization stage, and stage of organic manifestation, result in a long-lasting immunity preventing a second occurrence of the disease. The local infections of the intestine,'e.g. dysentery, cholera, salmonellal enteritis, dyspepsiacoli infections, etc. do not exhibit any progression in phase and result only in a weak antitoxic immunity. Considering the general theory that the disease itself develops the maximum antitoxic immunity to a reoccurrence of the disease, it is understandable that various injection vaccination processes developed for local intestinal infections are relatively inadequate.

This lack of prophylaxis against local intestinal infections is becoming of increasing importance. For example, bacterial dysentery and cholera, especially in subtropical and tropical climates, are endemically existent constantly, Whereas others are gaining in importance as severe medical problems, such as enteritis infectiosa salmonellosa, the dyspepsiacoli diseases, and the infections caused by several enterd-virusesKCoxsackie and ECHO viruses). Furthermore, the number of salmonellal infections in cattle has recently been increasing to a considerable extent. Heretofore,' n'o immunoprophylactic has been uncovered which is effective against all of these germs; even the cholera injection vaccination, although resulting in a milder form of the disease, cannot prevent the occurrence thereof.

-Consequently, an immunoprophylactic against enteritis-causing agents is indicated, and certainly urgently requiredin epidemic and disaster areas and even when traveling in areas having substandard hygienic conditions. Since,,inmost cases, the causative agent of the enteritis which will endanger the particular person remains unknown, it is desirable to effect immunization against all types ,of possible causative microorganisms. Similar considerations apply for an immunoprophylactic in connection with cattle; in this case, various severe breeding epidemics are caused, in particular, by different salmonellae introduced by imported feeds, and certain types of coli bacteria. Accordingly, a constant immunoprophylaxis 1s indicated in, case of domestic cattle.

However, there are more than 500 Salmonella types facultatively causing diseases in humans and animals; 22 types of dysentery bacteria pathogenic solely in the human; and a large, still unknown number of coli bacteria (at least types) pathogenic, in part to humans and, in part, to young animals. Accordingly, a combination vaccine against all possible enteritides would have to contain the antigen of at least about 700 types of causative agents, with the additional consideration that new types are constantly being discovered.

SUMMARY OF THE INVENTION Bearing the above considerations in mind, a principal object of this invention is to provide improved methods and compositions for effecting prophylactic immunization against local intestinal infections.

Upon further study of the specification and claims, other objects and advantages of the present invention will become apparent.

To achieve these objects, there is provided an oral polyvalent vaccine composition containing a synergistic mixture of different inactivated vaccine types specific against different groups of microorganisms.

The fact that the oral administration of a mixture of inactivated vaccines results in a satisfactory, functional immunity of the intestine against the different types of local infections is a surprising discovery. For example, it was discovered that mice were still capable of forming specific antibodies even 257 days after oral antigen administration.

This invention, therefore, exhibits a number of decisive advantages, viz.

The antigens, when ingested appear in the blood stream Within a few hours, since they are readily resorbed by the intestinal mucosa. In the intestinal mucosa, the antigens activate immunogenic cells which are locally detectable. Once the orally administered antigens have arrived in the blood, and thus in the reticuloendothelial system (RES), they initiate a general defense reaction in the body. Accordingly, after an oral vaccination, a satisfactory, functional, predominantly cellular immunity of the intestine is produced, as well as an immunological reaction of the entire body.

Numerous antigens can be combined without interfering with one another. Combinations of antigens proved compatible when ingested. Since the dosage for oral vaccines is practically unlimited, as many antigens as desired can be combined in sufliciently high concentrations. An oral vaccination can be repeated without any technical difliculties; accordingly, the body is not forced, as in parenteral vaccination, to react suddenly to a single influx of antigen of high concentration. Furthermore, the danger of allergic reactions, which is still very great in connection with injection vaccines, is substantially avoided when administering the antigen orally. Finally, this oral method is of great advantage for the prevention of epidemics. As experience has shown, voluntary participation in a vaccination drive is much higher when the vaccine is administered orally than when vaccinating by injection. Moreover, participation of the population to a higher degree is required in order to prevent the spreading of an infectious disease.

By oral vaccination, it is possible to employ considerably higher amounts of antigen, so that it is theoretically possible to accommodate about 700 antigens in the vaccine; however, in practice the combination of so many strains of bacteria is very difficult. It is actually unnecessary to employ so many strains in an oral vaccine. In contrast to the parenteral administration of antigen, the prophylactic effect is non-specific with respect to the causative agent and the subsequent infection. In addition to providing protection against the infection of the homologous strain of the bacterium, protection is also provided to a significant extent against related strains, although not entirely as effectively as by the strain-specific vaccination. Thus, for example, an oral vaccination with Salmonella typhimurium bacteria protects against an oral infection with Salmonella enteritidis, and vice versa (Zbl. f. Bak. I. Orig. 197, 368 [1965]). Accordingly, it would be sufiicient to combine, from each group of causative agents, only a few types of such agents in the vaccine. However, they would have to be used in uniformly high dosages, which again would require a total amount of antigen in the vaccine that is unfeasible and much too costly.

Thus, the key to the practical utilization of this invention is the unexpected discovery that after the oral administration of several antigens, the non-specific components of the antigen mixture functioned as potentiators by increasing the specific effect of the antigen mixture; in other words, a considerable synergistic efl'ect occurred. Thus, for example, a five-fold vaccine containing five different antigens in equal parts in such a total amount as ordinarily present in a single vaccine, afforded the same immunization protection as a corresponding single vaccine accordingly containing five times the amount of the homol- Ogous antigen.

The use of the vaccines of this invention, and the type and the amount of the antigens and their combination in such oral vaccines is dependent on the respective epidemiologic situation and the desired objective of the immunoprophylaxis. In disaster areas, (earthquakes, floods, and similar catastrophes), there will be incorporated, if possible, into the combination vaccine all the antigens of those disease-causing agents which are to be expected in the respective area, preferably salmonellae, shigellae, and dyspepsiacoli bacteria. In endemic cholera zones, antigens of the vibrions will also be incorporated into the vaccine. For travelers in subtropical and tropical areas, they will likewise be vaccinated with a combination of inactivated salmonellae, shigellae, and dyspepsiacoli bacteria.

It is another embodiment of this invention to administer the antigens of the last-mentioned types of bacteria for protecting infants in hospitals, homes or day nurseries against dyspepsiacoli infections.

Finally, to obtain an immunization of young calves against the dreaded calf dysentery, the administration of the antigens of the corresponding coli germs would be sufficient.

The preparation of the combined vaccine is dependent on its utilization. Normally, the microorganisms, inactivated according to conventional methods, are mixed in freeze-dried form and administered in the conventional pharmaceutical forms, such as, for example, drages, tablets, pills lozenges, syrups, or capsules; however, it is also possible to mix the antigens into liquids without any previous freeze-drying.

Drages, tablets, pills are also characterized by the inclusion of talc and/or carbohydrate carrier or builder or the like being preferably lactose, and/ or corn starch and/ or potato starch. For syrups and lozenges a sweetened 'vehicle is employed. The carrier is usually present in an amount of l-10,000 mg., preferably 1-5,000 mg.

The vaccines can be administered to all types of animals, e.g. mammals or avians. Suitably, for infants, small children and young cattle, the freeze-dried mixed antigen can be admixed with food. For purposes of a prophylactic immunization of adults, an oral application of respectively 50 mg. increments of a lyophilized mixed antigen, administered on five successive days, proved to be suitable; however, the daily dosage can also be increased without incurring any problems. In general, a daily dosage for animals ranging between 0.1 mg. to 10.0 mg., preferably 0.4 mg. to 1.3 mg. per kg. of body weight is preferred and the treatment can be administered for 3 to 20, preferably 4 to days. The preferred dosage range may be also expressed as 10 and 10 more preferably 10 inactivated germs per kg. of body weight.

The term polyvalen as used herein refers to a combination of different species of bacteria.

Without further elaboration, .it is believed that one skilled in the art, can, using the preceding description, utilize the present invention to its fullest extent; "The following preferred specific embodiments are,-therefore, to be construed as merely illustrative, and,v not limitative of the remainder of the disclosure in any way; whatsoever'i EXAMPLE 1 One-hundred white mice were orallyyaccinated,daily, ten times, on successive days, with a mbnovalent'vaifcine of a thermally inactivated dyspepsiacoli strain (No. 23 the total amount of germs administered being 7.5-10 germs per mouse. Another populationof- '100 mice were orally vaccinated with a triple vaccine containing. the same total number of germs but consisting, in thirds, of 2 (a) the same coli strain; l (b) a Salmonella typhimurium strain; and (c) a Shigella flexneri 2a-strain.

As a control, still another population of mice were not vaccinated. On the tenth day, after the last vaccination the 300 mice were simultaneouslycinfected with the live coli strain 2380.

Among the non-vaccinated control animals,;63% died; of the two vaccinated groups, only 29% died in each group. This means the triple vaccine unexpectedly had. the same satisfactory effect as the singlevaccine althougk it contained only one-third of the same homologous antigen. As a further check, the aforesaid monovalent vaccine! is diluted with an inert substance to one-third of the concentration, and in this case the mortality rate rises to about 51%, thereby proving the triple vaccine exhibits a-synergistic activity. 2

EXAMPLE 2 123 mice were orally vaccinated as in Example lqwith thermally inactivated dyspepsiacoli strain No. 2380;---247 mice received a quintuple vaccine of the same total number of germs, containing in equal parts: 1"

(a) the same coli strain; h I H (b) inactivated Salmonella typhimuriumj I (c) Salmonella schwartzengrund; (d) Salmonella blockley; and

(e) Shigella flexneri 2a bacteria.

l f Preparation of vaccines-.-

The strain of bacterium employed for the preparation of the vaccine (bacteria from thelgr'oupof the Exit bacteriaceae) is stored in theconventi onal "manneri laboratory. From the laboratory culture collectiomap liminary culture is inoculated in a liquid" med'ium and grown for about 8 hours at 37 C. 'lhis prelimitiary culture is then inoculated onto the s'urface'o f a conventional solid nutrient substrate disposed infcontainersihavir'fg'a large surface area. After breeding the cultureifo'r; 24'liour s on the solid substrate, the bacteria are removed float;- ing with physiological NaCl solutio'nQThe bacteriasuspension is centrifuged, the excess liquidis fcliscarded'l and the bacteria material is again takenjup' in' 'fresh"'physiological NaCl solution. The suspensieri'is centrifuged,and

the washing liquid is discarded. The washing process is normally repeated once. After the last washing step, the bacterial residue is again taken up in physiological NaCl solution or in distilled water and heated for about 20 minutes to 100 C. (inactivation of the live germs). The thus inactivated bacteria are separated from the liquid phase by centrifuging in the manner set forth above for the washing process, and lyophilized in accordance with any conventional process.

When conducting this method of preparation, x10 germs on a dry basis weigh approximately 1 mg.

EXAMPLE 4 Gelatine capsules with 50 mg. of lyophilized vaccine Ten grams each of:

(a) lyophilized germs of E. coli No. 2380;

(b) S. typhimurium; and (c) Sh. flexneri 2a,

all inactivated in accordance with Example 3, are mixed homogeneously and then filled, in amounts of 50 mg., into hard gelatin (plug-together type) capsules.

EXAMPLE 5 Tablets with 50 mg. of lyophilized vaccine Composition for one tablet: 50 mg. of lyophilized, homogeneously mixed germs, inactivated according to Example 3, of equal parts of:

(a) E. coli No. 2380; (b) S. typhimurium; and (c) Sh. flexneria 2a;

Immunizing milk having a vaccine content of '50 mg./100 ml. of liquid Five grams, respectively, of inactivated germs of E. coli No. 2380, S. typhimurium and Sh. flexneri 2a are suspended in 301. of pasteurized milk, and filled into 100 ml. bottles.

EXAMPLE 7 Lozenges for buccal application having mg. of lyophilized vaccine Composition for one lozenge:

. Mg. Inactivated, lyophilized germs of E. coli 2380 5.00 Inactivated, lyophilized germs of S. typhz'murium 5.00 Inactivated, lyophilized germs of Sh. flexneri 2a 5.00 Powdered sugar 300.00 Licorice juice 130.00 Licorice powder 25.00 Gum arabic 30.00

6 EXAMPLE 8 Tablets with 50 mg. of lyophilized vaccine Composition for one tablet: 50 mg. of lyophilized, homogeneously mixed germs, inactivated according to Example 3, of

(a) 20% of E. coli No. 2380;

(b) 50% of S. typhimurium; and (c) 30% of Sh. flexneria 2a;

What is claimed is:

1. A polyvalent vaccine composition in oral dosage form for effecting prophylactic protection against a plurality of local intestinal bacterial infections, said composition comprising an effective amount of a synergistic mixture of the inactivated form of a plurality of virulent species of bacteria selected from a plurality of the members of the group consisting of E. Coli, Salmonella and Shigella, each of said species being present in an amount which separately provides incomplete prophylactic protection against infection caused by the virulent form of that species.

2. A vaccine composition as defined by claim 1 wherein the composition is in the form of solid compositions in unit dosage form of a capsule.

3. A vaccine composition as defined by claim 1 wherein the composition is in the form of a lozenge.

4. A vaccine composition as defined by claim 1 wherein the composition is in the form of a syrup.

5. A vaccine composition as defined by claim 1 wherein the composition is in the form of a suspension.

6. A vaccine composition as defined by claim 1 wherein the composition is in the form of solid compositions in unit dosage form containing talc as a binder.

7. A vaccine composition as defined by claim 1 wherein the composition is in the form of solid compositions in unit dosage form containing a carbohydrate as a binder.

8. A vaccine composition as defined by claim 1 wherein said mixture comprises inactivated species of bacteria from each of the groups:

(a) dyspepsiacoli,

(b) Salmonella, and

(c) Shigella.

9. A composition according to claim 8 comprising S. typhimurium and Sh. flexneria.

10. A composition according to claim 8 wherein the species are present in about equal amounts.

References Cited FOREIGN PATENTS 417,382 9/1934 Great Britain 424-92 44,288 10/1938 Netherlands 42492 RICHARD L. HUFF, Primary Examiner 

